The Evolution of Cell Culture Systems to Study Hepatitis B Virus Pathogenesis and Antiviral Susceptibility

Abstract

The global burden of hepatitis B virus (HBV) remains high, with ongoing concerted efforts to eliminate viral hepatitis as a public health concern by 2030. The absence of curative treatment against HBV makes it an active area of research to further study HBV pathogenesis. In vitro cell culture systems are essential in exploration of molecular mechanisms for HBV propagation and the development of therapeutic targets for antiviral agents. The lack of an efficient cell culture system is one of the challenges limiting the development and study of novel antiviral strategies for HBV infection. However, the evolution of cell culture systems to study HBV pathogenesis and treatment susceptibility in vitro has made a significant contribution to public health. The currently available cell culture systems to grow HBV have their advantages and limitations, requiring further optimization. The discovery of sodium taurocholate co-transporting polypeptide (NTCP) as a receptor for HBV was a major breakthrough for the development of a robust cell model, allowing the study of de novo HBV infection through NTCP expression in the HepG2 hepatoma cell line. This review is aimed at highlighting the evolution of cell culture systems to study HBV pathogenesis and in vitro treatment susceptibility.

Keywords: HepG2-NTCP cells; cell culture systems; hepatitis B virus (HBV).

Figure 1

Structural representation of infectious HBV virions. HBsAg S, M, and L surface proteins on the lipid envelope. The lipid envelope surrounds the nucleocapsid (containing a relaxed circular DNA (rcDNA) and the viral DNA polymerase. Created in BioRender. Xaba, L. (2025) https://BioRender.com/i0t9boo (accessed on 18 July 2025).

Figure 2

Schematic illustration of HBV covalently closed circular DNA (cccDNA) fate in HBV-infected PHH and HBV spread in HepG2-NTCP cells [57,58]. (A) Proliferation of HBV-infected hepatocytes results in the loss of cccDNA; this shows how individual cccDNA molecules are distributed during cell division [58]. Red-shaded squares represent cells infected with active HBV replication and cccDNA is represented by the infinity symbol (∞). (B) Schematic representation of experimental designs showing fate of HBV in two co-culture systems of HBV donor (red squared with green circles) and acceptor cells (white squares with white circles) in the presence of PEG [57]. Red squares with white circles are infected acceptor cells due to spread. PHH: primary human hepatocyte; NTCP: sodium taurocholate co-transporting polypeptide (adapted with permission from Professor Maura Dandri).

Figure 3

HBV cell culture systems in chronological order of development.

Figure 4

Diagrammatic representation of HBV entry into HepG2-NTCP cells via NTCP. HBV interacts with the HSPG on the cell surface and binds to the specific receptor NTCP on the HepG2-NTCP cell, and then proceeds to enter the HepG2-NTCP cell. HSPG: heparan sulphate proteoglycan; NTCP: Na+-taurocholate co-transporting polypeptide; and cccDNA: covalently closed circular DNA [79]. Created in BioRender. Mthethwa, L. (2025) https://BioRender.com/r09j085 (accessed on 18 July 2025).

Figure 5

Diagrammatic representation showing where the inhibitors are acting in the replication cycle of HBV. Created in BioRender. Mthethwa, L. (2025) https://BioRender.com/r09j085 (accessed on 18 July 2025) and edited in Microsoft PowerPoint.